The present invention relates to compositions and kits for use in enzyme-prodrug therapy, in which the enzyme is conjugated to a carrier.
The chemotherapy of neoplastic disease is often compromised by adverse systemic toxicity which limits the dose of drug that can be administered, or is limited by the appearance of multidrug resistance. Various strategies have been explored to improve drug targeting and to increase drug concentration in the tumour to a level that should overcome clinically relevant drug resistance.
Prodrugs have been used for many years in medicine to treat a variety of disorders. They can offer improved solubility, improved pharmacokinetics and tissue distribution, avoidance of unfavourable metabolism, selective organ effects and specific tumour toxicity. In order to use prodrugs as anticancer agents, the tumour must have a high level of the enzyme that activates the prodrug, or a foreign enzyme must be delivered, and no activating enzyme must be present in normal tissues. The prodrug must be innocuous and pharmacodynamically inert and be a substrate for the enzyme with favourable Km and Vmax values.
The concepts of Antibody Directed Enzyme Prodrug Therapy (ADEPT) (refs. 1,2) and Gene/Viral Directed Enzyme Prodrug Therapy (G/VDEPT) (ref. 3) using either antibody conjugates or a retroviral vector to deliver an enzyme to a tumour, are already well established. In ADEPT, a foreign enzyme which metabolises substrates not normally metabolised by mammalian cells is linked chemically to a tumour-specific or tumour-associated antibody; the antibody conjugate is injected intravenously and binds strongly to the tumour by recognising the tumour-associated antigen. In VDEPT, the retroviral vector is designed to carry a gene expressing a preselected enzyme that can be delivered selectively to tumour or be under the control of a tumour-specific promoter. The foreign enzyme is then used to activate a carefully designed low molecular weight prodrug. Animal models and pilot human studies have proven that it is possible to deliver selectively to a solid tumour an activating enzyme such as carboxypeptidase G2, penicillin amidase, xcex2-lactamase, xcex2-glucuronidase, cytosine deaminase, nitroreductase or alkaline phosphatase (refs. 4,5).
Both approaches have a number of inherent limitations. In the case of ADEPT, these include the immunogenicity of the antibody-enzyme conjugate, the need to tailor each conjugate to target antigen present on the tumour, the difficulty in optimisation of the dosing schedule, and the need to use a clearing antibody in the case of ADEPT (ref. 6). In the case of VDEPT, there are the inherent dangers associated with a viral vector, the potential lack of specificity of enzyme expression in the tumour due to problems of delivering the gene specifically to cancer cells, and the difficulty of evaluating the duration and reproducibility of enzyme expression on a patient basis, leading to difficulties in optimising the schedule of prodrug follow-up.
In recent years, there has been a great deal of investigation of polymers as carriers of anticancer drugs (reviewed in ref. 7). The basis for much of this work is that attachment of toxic drugs to high molecular weight carriers can lead to reduction in systemic toxicity, longer retention time in the body, alterations in biological distribution, improvements in therapeutic efficacy and site-specific passive capture through the enhanced permeability and retention (EPR) effect. The EPR effect results from enhanced permeability of macromolecules or small particles within the tumour neovasculature, due to the leakiness of its discontinuous endothelium. In addition to the tumour angiogenesis (hypervasculature) and irregular and incompleteness of vascular networks, the attendant lack of lymphatic drainage promotes accumulation of macromolecules that extravasate (ref. 8). This effect is observed in many solid tumours for macromolecular agents and lipids. The enhanced vascular permeability will support the great demand of nutrients and oxygen for the rapid growth of the tumour. Unless specifically addressed for tumour cell uptake by receptor-medicated endocytosis, polymers entering the intratumoural environment are taken up relatively slowly by fluid-phase pinocytosis.
Many polymer-based anticancer agents have now entered the clinic or are passing through clinical trials; each has proven the concept compared to the native drug. For instance, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugates have already shown promise in early clinical trial (refs. 9,10). Moreover, residual HPMA copolymer conjugate which does not permeate in to the tumour but remains in the circulation is rapidly excreted, giving a high tumour:blood ratio (ref. 11).
In most cases, release of active anticancer drug from the polymer support is mediated by simple aqueous hydrolysis or by proteolytic or esterase enzymes (refs. 12,13). Since the conditions for these reactions are not necessarily confined to tumour tissues, some non-specific drug release is inevitable.
Conjugation of water-soluble polymers to pharmacologically-active proteins such as enzymes, toxins, immunoglobulins, cytoxins or allergens can be used to reduce the proteolytic degradation of such proteins, improve their biological efficacy and prolong plasma elimination, as well as reduce protein immunogenicity. For instance, an HPMA copolymer-asparaginase conjugate has been used to treat leukaemia. The conjugation of the enzyme to the polymer has been shown to increase the circulation time of the enzyme, where its activity in depriving leukaemic cells of asparagine is useful.
According to the present invention, a product or kit comprises two components, i.e. two pharmaceutical compositions that are arranged or otherwise adapted for sequential administration to a human or animal. The first component is an enzyme conjugate, e.g. a composition that comprises a pharmaceutically-acceptable excipient and an enzyme conjugate. The enzyme conjugate may consist of an enzyme covalently bound to a polymeric or other carrier such that the enzyme conjugate retains its enzyme activity. The second component is a prodrug, e.g. a composition that comprises a pharmaceutically-acceptable excipient and a prodrug. The prodrug is typically substantially inactive (in terms of drug activity) but capable of being activated by the enzyme.
It is to be noted that unless the context specifically refers to the order of administration of the product, reference herein to xe2x80x9cfirstxe2x80x9d and xe2x80x9csecondxe2x80x9d compositions does not imply any specific order of administration.
The two components of the kit are used in a treatment regimen similar to ADEPT, except that the enzyme conjugate is administered in place of the antibody-enzyme conjugate. One significant difference is that the two components can be administered, in use of the present invention, in either order. For instance, prodrug-containing composition may be given first. The present invention can avoid the problems with ADEPT, resulting from the immunogenicity of the antibody-enzyme conjugate. Furthermore, although a polymer carrier is not specifically targeted to antigenic sites of the tumour cell surface, it is nevertheless expected to preferentially accumulate in solid tumours through the EPR effect. It has been observed that ADEPT is surprisingly non-specific in vivo, and it is believed that the polymerenzyme conjugate is likely to exhibit preferential accumulation as significant as that exhibited in ADEPT.